Many enzymes in cells are modified covalently as a regulatory mechanism. For example, protein phosphorylation is a modification central to the regulation of metabolism and other cell functions. Another modification is ADP-ribosylation, best known from the modification of G-proteins by the bacterial toxins cholera toxin and pertussis toxin, which are involved in the pathogenesis of cholera and whooping cough, respectively. Previous work demonstrated the presence of cellular enzymes that catalyze reactions similar to those catalyzed by the bacterial toxins; it is important to understand how ADP-ribosylation catalyzed by endogenous enzymes may regulate animal cell function. In the current work, ADP-ribosylation was characterized in cell-free systems. Several proteins were ADP-ribosylated during incubation of brain supernatant with adenine-labelled NAD. One modified protein was purified and identified as aldehyde dehydrogenase (ALDH). The interactions of NAD and ADP-ribose, a reactive metabolite of NAD, with pure ALDH were investigated. ALDH was covalently modified by ADP-ribose stoichiometrically, accompanied by nearly complete inhibition of enzyme activity. ADP-ribosylation and inhibition were reduced by the presence of NAD, indicating that ADP-ribose may be modifying ALDH at the active site. The chemical properties of the ALDH-ADP-ribose linkage indicated that the modification may be at a cysteine residue. Other related enzymes, including other dehydrogenases with a catalytic cysteine residue such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were labelled to a much lesser extent with ADP-ribose. In conjunction with our previous work demonstrating nonenzymatic ADP-ribosylation of free cysteine, the present identification of stoichiometric ADP-ribosylation of proteins on cysteine residues indicates the necessity of considering these nonenzymatic reactions during the study of endogenous ADP-ribosylation.